Viruses create cellular conditions conducive to their own replication by harnessing or inactivating cellular machinery. Since viruses possess small genomes that do not encode all the factors required for DNA replication, they rely on host cell proteins to propagate their genomes. Active recruitment of cellular proteins to viral replication compartments functions to promote virus replication. In contrast, viral early proteins target antiviral cellular DNA sensors and transcriptional repressors to prevent their access to viral genomes. Identifying host factors that facilitate or limit virus DNA replication has been hampered by the lack of technologies, and only a limited number of cellular proteins have been shown to affect virus replication. Our long-term goal is to understand how cells respond to virus genomes, and to define ways that early viral proteins overcome cellular restrictions to promote virus replication. In this application we use Adenovirus infection as a model system, and employ a novel technology to identify cellular proteins that associate with replicating viral DNA genomes. Our central hypothesis is that viral proteins selectively recruit cellular DNA replication/repair factors onto viral genomes to function with viral-encoded replication factors, while also targeting specific host antiviral factors to prevent their access to viral genomes. We propose that viral early E1b and E4 gene products overcome host responses and achieve their pleiotropic functions by influencing the cellular proteins recruited to virus genomes. These viral early proteins facilitate viral DNA replication, RNA processing, protein expression, and progeny production, but their substrates are currently poorly understood. We employ novel proteomic approaches to identify host factors that interact with these viral early proteins, and determine how they impact the landscape of host factors that recognize viral DNA. Guided by strong preliminary data, we propose three Specific Aims to identify host factors recruited or inactivated by viral early proteins and determine their impact on virus DNA replication. Aim 1: To determine how host factors associated with replicating viral genomes impact infection. Aim 2: To define cellular responses across the Ad family. Aim 3: To determine how E1b55K interacting proteins impact virus infection. These studies employ innovative approaches to provide a comprehensive view of cellular responses to foreign DNA genomes replicating in the host cell nucleus. We anticipate that our results will identify proteins recruited to aid viral replication, as well as wys viral proteins manipulate host responses. Identifying cellular proteins commonly exploited for viral DNA replication across different viruses will suggest potential targets for development of novel broadly acting antiviral therapeutics. Our studies provide insights into cellular sensors of foreign DNA, and these will guide development of improved gene delivery vectors, viral oncolytics, and antiviral therapies.